Most organisms that experience daily light/dark or temperature cycles have a molecular oscillator called the circadian clock. The circadian clock is important for coordinating daily physiological activities and processes with specific times of day. Importantly, the circadian clock provides fitness advantage when it functions correctly. The molecular and biochemical mechanisms of the circadian clock are not well understood, but it is clear that the basic architecture of circadian clocks is similar in eukaryotes. The objective of this proposal is to use Arabidopsis as a model to study eukaryotic circadian clocks. Specifically, the biochemical role of the central clock component, TIMING OF CAB EXPRESSION 1 (TOC1), remains an outstanding question in the Arabidopsis circadian clock field. In this proposal, the recently discovered TOC1-interacting transcription factor, CCA1 PROMOTER HIKING EXPEDITION 1 (CHE1) will be used to determine the biochemical role of TOC1. This work will connect the core components of the Arabidopsis circadian clock and will increase our understanding of eukaryotic clock architecture. Specific Aims 1. Determine the roles of TOC1 and ZTL in CHE regulation. The interaction of TOC1 and CHE is the first biochemical link between TOC1 and control of CCA1 expression. Biochemistry, transient transcription assays, and genetics will be used to determine the role of TOC1 and ZTL in regulation of CHE function. 2. Discovery of transcriptional complexes that regulate LHY and CCA1 expression. A library of all transcription factors from Arabidopsis has been created in the Kay lab. This library will be screened by yeast two-hybrid to find transcription factors that interact with TOC1 or ZTL. A streamlined set of in vivo biochemical and molecular techniques will be used to prioritize and categorize the transcription factors that interact with TOC1/ZTL but also regulate LHY and CCA1 expression. Public Health Relevance: Circadian clock defects are known to result in a range of human health related disorders, and clock components are involved in cancer progression and cancer attenuates clock function. The results of this study will increase our understanding of circadian clock architecture in eukaryotes and thus will be relevant to the study and treatment of clock related diseases.